Proteins are responsible for a majority of the cellular functions such as molecular recognition (for example in the immune system), signaling pathways (hormones), the transport of metabolites and nutrients and the catalysis of biochemical reactions (enzymes).
The function of proteins results from their three-dimensional structure, that is to say how the amino acids of the polypeptide chain are arranged relative to each other in space. It is usually only in its folded state (native state) that a protein can exert its biological activity.
Whereas most proteins have a primary structure (amino acid sequence), a secondary structure (alpha-helices and beta-sheets), and a tertiary structure (three-dimensional), protein oligomers have an additional level called the quaternary structure that is part of the three-dimensional structure. Oligomers are complexes of several polypeptides. They can contain several copies of an identical protein referred to as a sub-unit and are referred to as homo-oligomers, or they may consist of more than one type of protein sub-unit, in which case they are referred to as hetero-oligomers. Hemoglobin, the oxygen carrier in blood, is an example of a protein containing identical subunits. Nitrogenase, the microbial enzyme responsible for the reduction of nitrogen gas to ammonia, is an example of a protein containing non-identical sub-units.
Numerous recombinant proteins of interest are oligomeric in nature, for example antibodies, many transmembrane proteins such as transmembrane receptors, porins, viral surface antigens, heat shock proteins, viral capsid proteins, ferritin, insulin, many enzymes such as glutathione peroxidase, catalase or superoxide dismutase, collagen and many others.
For instance, influenza virus haemagglutinin (HA) is a homotrimeric glycoprotein on the surface of the virus which is responsible for interaction of the virus with host cell receptors. The three-dimensional structure of HA is described in detail in Nature, 289, 366-373 (1981). Protective immune responses induced by vaccination against influenza virus are primarily directed to the viral HA protein. Recombinant HA protein (rHA) represents therefore an interesting antigen for the development of influenza vaccines.
Another oligomeric antigen of interest is the Invasion Plasmid Antigen D (IpaD) protein of Shigella that was found to form either pentamers, or in the presence of IpaB, tetramers, at the needle tip of the bacteria (Cheung et al., Molecular Microbiology, 95(1), 31-50 (2015)).
A further oligomeric antigen of interest is the Membrane expression of Ipa H (MxiH) protein of Shigella that was found to form a helical assembly of subunits that produces the Shigella needle (Cordes et al., The Journal of Biological Chemistry, 278(19), 17103-17107 (2003)).
One of the challenges in the recombinant protein field is that recombinant proteins do not always have the same three-dimensional conformation as the native protein. Yet the function of proteins often results from their three-dimensional structure.
Similarly, in respect of oligomers, if the recombinant protein does not keep the quaternary structure of the native protein, the function of the recombinant protein may be altered or suppressed.
For instance, William C. Weldon et al., in Plos One, 5(9), e12466 (2010), showed that poor trimerization of a recombinant influenza haemagglutinin could play a role in its low immunogenicity.
There is therefore a need to produce recombinant proteins which better retain the oligomeric structure and desired biological function of the native protein.
Chih-Jen Wei et al., in Journal of Virology, 82(13), 6200-6208 (2008), describe the trimerization of influenza rHA using the foldon sequence of the T4 phage.